Chondrocyte therapeutic delivery system

ABSTRACT

Systems and methods for modifying the environment of target cell using genetically altered chondrocytes are provided. The genetically engineered chondrocytes can be used to express a therapeutic agent in a subject, including in an environment typically associated with chondrocytes and in an environment not typically associated with chondrocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/328,083 filed onDec. 4, 2008 and entitled “Chondrocyte Therapeutic Delivery System,”which claims priority to and is a continuation-in-part of U.S. patentapplication Ser. No. 10/657,516 filed on Sep. 8, 2003, now U.S. Pat. No.7,897,384, and U.S. patent application Ser. No. 12/123,650 filed on May20, 2008, which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/941,326 filed Jun. 1, 2007. All related applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the treatment or amelioration of disorders ordiseases by the delivery of therapeutic agents(s) produced viagenetically-altered chondrocytes chondrocytes to various treatmentsites.

BACKGROUND OF THE INVENTION

The use of various types of biologically active cells as components ofimplanted devices for the purpose of delivering bioactive agentsproduced by said cells is well known in the art. Various attempts todevise methods for enhancing the survivability of such implanted cellshave ultimately fallen short and require either the vascularization ofthe implant or a maximum diffusion distance of about 2-3 mm in order toprovide nutrients to the cells. These various types of cells includefibroblasts, myoblasts, stem cells, progenitor cells, maturedifferentiated tissue cells, and undifferentiated cells.

Chondrocytes offer several unique advantages as vehicles for expressingtherapeutic agents over other cell types. For example, chondrocytes donot require vascular support, and therefore can readily be used inenvironments that have a reduced, or non-existent vascularizationsystem. Furthermore, chondrocytes are able to survive in harsh in vivoenvironments, including low pH and low oxygen surroundings. In addition,there is a reduced likelihood of malignancy due to the anti-angiogenicproperties of normal chondrocytes. Chondrocytes also possess an immuneprivileged property which reduces immune rejection of co-implantedallogenic or xenogenic tissue. Furthermore, chondrocytes are more easilyscalable compared to other normal untransformed cell strains.

Chondrocytes are typically involved in cartilage repair. Cartilage is astructural support tissue that is found in the body in three mainvarieties. Hyaline, or articular cartilage, helps dissipate loads injoints. In articular cartilage, chondrocytes are encapsulated in awoven, mesh-like matrix of type II collagen and proteoglycans. Elasticcartilage provides flexible support to external structures, and iscomposed of chondrocytes embedded in a matrix of collagen and elasticfibers. Fibrocartilage aids in transferring loads between tendons andbone. It consists of an outer layer of collagen and fibroblasts thatsupport and inner layer of chondrocytes that make type II collagenfibers.

To date chondrocytes have been used to correct or repair cartilaginousdefects. For example, by placing chondrocytes into hydrogels andinjecting the chondrocytes into a cartilage defective region. Thechondrocytes are used to express matrix proteins required for cartilagerepair. Although chondrocytes have been cultured and placed intosubstrates such as hydrogels, for treatment, or repair of cartilage orbone defects, there is no teaching in the art that chondrocytes can beused to express therapeutic agents for the treatment of pathologies orinjuries other than cartilage tissue.

However, there remains a need for devices and methods capable ofdelivering a large volume of such genetically-altered chondrocytes tovarious treatment sites not typically associated with chondroctyes.

SUMMARY OF THE INVENTION

The present invention provides systems and methods for the delivery ofgenetically altered chondrocytes to various treatment sites to expresstherapeutic agents. The systems and methods of the invention areparticularly conducive to the use of chondrocytes for expressing atherapeutic agent in localized environments that are not typicallyassociated with chondrocytes. Specifically, the genetically alteredchondrocytes can be used to express therapeutic agents, such as proteinsor antibodies, into localized environments such as the central nervoussystem (e.g., the brain or spinal cord) or into solid organs (e.g.,heart, liver or kidney). This type of localized delivery and expressionof the therapeutic agent provides a localized concentration of thetherapeutic agent in a particular region, without the necessary sideeffects that result from systemic administration of the therapeuticagent. In addition, if it is desirable to deliver a therapeutic agentsystemically, this can still be done by placing the cells near avascular structure, including subcutaneously.

Chondrocytes offer several unique advantages as vehicles for expressingtherapeutic agents over other cell types. For example, chondrocytes donot require vascular support, therefore can readily be used inenvironments that have a reduced, or non-existent vascularizationsystem. Furthermore, chondrocytes are able to survive in harsh in vivoenvironments, including ischemic tissue, as well as low pH and lowoxygen surroundings. In addition, there is a reduced likelihood ofmalignancy due to the anti-angiogenicity properties of normalchondrocytes. Chondrocytes also possess an immune privileged propertywhich reduces immune rejection of co-implanted allogenic or xenogenictissue. Furthermore, chondrocytes are more easily scalable compared toother normal untransformed cell strains.

The system includes a housing configured to allow passage of atherapeutic agent and at least one genetically altered chondrocyteretained within the housing. As discussed in more detail below, thegenetically altered chondrocyte is modified to express a therapeuticagent. The housing defines an internal cell chamber configured to retaingenetically altered chondrocytes. While preventing passage of thesechondrocytes, the housing can be configured via a number of exit ports(e.g., semi-permeable membranes) to allow for the controlled release oftherapeutic agents produced by these entrapped cells. As will be shownin detail below, the cell chamber can be designed such that the core ofthe chamber can reside a significant distance from the outer wall of thedevice (e.g., greater than at least about 1.5 mm). Such a large-scalecell chamber can be utilized in light of the chondrocytes' ability toremain viable even when separated from an external nutrient supply. Asshown below, the use of such large-scale delivery systems cansignificantly increase the amount of therapeutic agent(s) capable ofbeing produced and delivered per delivery device. Various aspects of thesystem and method are summarized below.

In one aspect, a therapeutic system is provided which includes a housinghaving a cell chamber configured to retain a genetically alteredchondrocyte. For example, the cell chamber can have a length and adiameter wherein each the length and the diameter are at least about 3mm in dimension. These dimensions provide a device capable of producingand delivering a large amount of therapeutic agent to the treatmentsite. Additionally, a portion of the housing can be configured to allowfor passage of the therapeutic agent(s) produced via the chondrocytesfrom the cell chamber to the treatment site. For example, the housingcan include at least one semi-permeable membrane capable of allowingrelease of the agents. The membrane can be limited to the distal end ofthe housing, or multiple membranes can be positioned at variouslocations along the housing (e.g., extending along each side of thehousing). Optionally, the housing, or at least a portion thereof, can bemade of a porous metal or ceramic material, such that the cells areretained within the housing but proteins are able to pass through thepores.

The housing can be configured to accommodate the desired mode ofdelivery, treatment site, type of therapeutic agent to be produced, etc.For example, the housing can include a rigid material, a non-rigidmaterial, or any combination thereof. Further, the distal end of thedevice can include a pointed (or tapered) section configured tofacilitate delivery of the device.

The housing can be configured in various manners to allow for delivery(or replenishment) of the chondrocytes to the delivery device. Forexample, the system can include a cap which can removably couple to anend of the housing such that removing the cap can allow for introductionof the chondrocytes to the cell chamber. In other embodiments, thehousing can include an injection port (e.g., a rubber septum) capable ofallowing for injection of additional cells via a syringe. In theseembodiment, the chondrocytes can be replenished to the device while thedevice remains at the surgical site thereby significantly increasingefficiency and safety.

In other embodiments, the therapeutic system can include an attachmentelement (e.g., a suture) coupled to the housing wherein the attachmentelement is configured to secure the device to tissue/bone at thetreatment site. In other embodiments, a radiopaque marker can bedisposed on or in the container so as to facilitate positioning and/orlocating the housing.

In other embodiments, the housing can include numerous additionalchambers so as to enhance efficiency. For example, in addition to thecell chamber, the housing can include an expandable chamber configuredto retain a water swellable material wherein the compartments areseparated by a piston. In such an embodiment, the introduction of waterinto the expandable chamber expands the water expandable materialthereby forcing the piston to move in a distal direction, and thusforcing an amount of therapeutic agent out of a distal exit port of thecell chamber. Water can be added to the expandable chamber by any numberof techniques. For example, the expandable chamber can include anosmotic membrane configured to allow for delivery of water.

In other embodiments, the housing can include an auxiliary fluid chamberpositioned between the cell chamber and the expandable chamber. Similarto the embodiment described above, the expandable chamber can include awater swellable material capable of driving a piston in a distaldirection upon the introduction of water. However, as opposed todirectly acting on the cell chamber, in this embodiment, the piston actson an auxiliary fluid retained within the auxiliary fluid chamber. Aswill be discussed, the auxiliary fluid (which can include cellnutrients, etc.) can be separated from the cell chamber via asemi-permeable membrane. As such, as the piston moves distally,auxiliary fluid is forced through the semi-permeable membrane and intocommunication with the condrocytes. Ultimately, the desired therapeuticagent(s) are forced out of an exit port (formed, for example, by anothersemi-permeable membrane) of the cell chamber, and to the treatment site.

In a further aspect, the housing can include a fluid pump and valvesystem configured to provide a pressurized flow of an auxiliary fluid tothe housing from an external reservoir. Additionally, the pump and valvesystem can provide a continuous outflow of desired therapeutics from thehousing to the treatment site. In some embodiments, the pump can be usedto remove cells from the housing, and also recharge the container withfresh cells. Also, the pump can be used to infuse the cell chamber ofthe container with various toxic agents in order to deactive the cellsupon completion of the therapeutic regimen.

In some embodiments, the exit port of the housing can be incommunication with a delivery tube capable of transporting thetherapeutic agents from the cell chamber to one or more distanttreatment locations. For example, the delivery tube can include numerousbranches so as to allow for delivery of therapeutics along each branchand ultimately to various distinct locations.

The genetically altered chondrocyte can be used to deliver and expresstherapeutic agents in an environment that requires modification with thetherapeutic agent. In one embodiment, the genetically alteredchondrocytes can be used to express a therapeutic agent in anenvironment associated with chondrocytes, such as the cartilage,tendons, joints, and bones. When the genetically engineered chondrocytesare used to express therapeutic agents in environments associated withchondrocytes, they are used for the purpose expressing the therapeuticagent, they do not become part of the structural component of theenvironment, i.e., they are not used as a component of a tissueengineered construct to repair or replace a damaged cartilage, tendon,joint, or bone. In another embodiment, the genetically alteredchondrocytes are used to produce a therapeutic agent at an ectopic site,that is, in an environment that is not typically associated withchondrocytes such as the brain, or solid organs (e.g., heart, kidney,liver). Other atypical chondrocyte environments include an aqueousenvironment such as blood, and plasma.

The genetically altered chondrocytes can be used to treat or amelioratea number of disorders by modifying the cell associated with the disorderby expressing the therapeutic agent. The cells associated with adisorder include disorders such as a blood disorder; a cardiovasculardisorder; an endocrine disorder; an autoimmune disorder; a neurologicaldisorder; a skin disorder; bone disorder (osteoporosis); a fertilitydisorder; a metabolic disorder or uncontrolled growth disorder such ascancer. In other embodiments, the genetically altered chondrocytes canalso be used to address conditions that are not associated with adisorder, for example, to express a therapeutic agent such as hormonesfor birth control and reproduction.

Accordingly, in one aspect, the invention pertains to a method ofmodifying the environment of a target cell associated with a disorder byusing a genetically altered chondrocyte to express a therapeutic agent.A housing is provided that is configured to retain the chondrocyte andthe housing and genetically altered chondrocyte are delivered to theenvironment of a target cell associated with a disorder. The geneticallyaltered chondrocyte expresses the therapeutic agent to modify theenvironment surrounding the target cell.

In one embodiment, the target cell associated with the disorder can be acell in an atypical chondrocyte environment such as an organ selectedfrom the group consisting of the brain, heart, liver, kidney,gastro-intestinal tract, spleen, smooth muscles, skeletal muscles, eye,ganglions, lungs, gonads, and pancreas. The atypical chondrocyteenvironment can also be an aqueous environment selected from the groupconsisting of blood, plasma, the vitreous humor of the eye, spinalfluid, and the like. The genetically altered chondrocyte can bedelivered to the spleen, the bone marrow, a ganglion, the peritonealcavity or any other well vascularized tissue for release into the bloodor plasma. In another embodiment, the cell associated with a disorder isin a typical chondrocyte environment such as a bone, tendon andcartilage.

The housing can be configured to accommodate the desired mode ofdelivery, treatment site, type of therapeutic agent to be produced, etc.For example, the housing can include a rigid material, a non-rigidmaterial, or any combination thereof. Further, a portion of the housingcan be configured to allow for passage of the therapeutic agent(s)produced via the chondrocytes from the housing to the treatment site.For example, the housing can include at least one semi-permeablemembrane capable of allowing release of the agents

The target cell associated with a disorder can be a cell selected fromthe group consisting of a cell associated with a blood disorder, a cellassociated with a cardiovascular disorder, a cell associated with anendocrine disorder, a cell associated with an autoimmune disorder, acell associated with a neurological disorder, a cell associated with askin disorder, a cell associated with fertility disorder, and cellassociated with reproduction.

Accordingly, in another aspect, the invention pertains to a method forameliorating a disorder in a subject by implanting a genetically alteredchondrocyte retained within a housing into a target region of thesubject. The genetically altered chondrocyte expresses the therapeuticagent at a level sufficient to ameliorate the disorder. As discussedabove, the housing can include a rigid material, a non-rigid material,or any combination thereof. Further, a portion of the housing can beconfigured to allow for passage of the therapeutic agent(s) produced viathe chondrocytes from the housing to the target region. For example, thehousing can include at least one semi-permeable membrane capable ofallowing release of the agents.

As will be discussed in more detail below, in one embodiment, thegenetically altered chondrocyte is not structurally functional in thetarget region or an environment surrounding the target region. In oneembodiment, the target region is atypical chondrocyte environment suchas an organ selected from the group consisting of the brain, heart,liver, kidney, gastro-intestinal tract, spleen, smooth muscles, skeletalmuscles, eye, skin, ganglions, lungs, gonads, and pancreas. The atypicalchondrocyte environment can also be an aqueous environment selected fromthe group consisting of blood, plasma, the vitreous humor of the eye,spinal fluid, and the like. The genetically altered chondrocyte can bedelivered to the spleen, the bone marrow, a ganglion, the peritonealcavity or any other well vascularized tissue for release into the bloodor plasma. In another embodiment, the cell associated with a disorder isin a typical chondrocyte environment such as a bone, tendon andcartilage.

The injury to be ameliorated can be a wound, a bone defect, a cartilagedefect, a skin wound, and a torn ligament. The disorder that can beameliorated can be a blood disorder, an autoimmune disorder, a hormonaldisorder, an anti-inflammatory disorder, a fertility disorder, and anneurodegenerative disorder.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a side view of an exemplary embodiment of a delivery deviceprovided herein;

FIG. 1B is an alternative embodiment of the device of FIG. 1A;

FIG. 1C is a cross-sectional view of the embodiment of FIG. 1A takenalong line C-C;

FIG. 2A is side view of another exemplary embodiment of the deliverydevice;

FIG. 2B is a side view of an alternative embodiment of the deliverydevice of FIG. 2A;

FIG. 2C is a side-view of another alternative embodiment of the deliverydevice of FIG. 2A;

FIG. 3 is a top view of another embodiment of the delivery device;

FIG. 4A is a cross-sectional view of another exemplary embodiment of thedelivery device wherein a piston element is in a first position;

FIG. 4B is a cross-sectional view of the device of FIG. 4A wherein thepiston is in a second position;

FIG. 5A is a cross-sectional view of another exemplary embodiment of thedelivery device wherein a piston is in a first position;

FIG. 5B is a cross-sectional view of the device of FIG. 5A wherein thepiston is in a second position;

FIG. 5C is a cross-sectional view of an alternative embodiment of thedevice of FIG. 5A;

FIG. 6A is a cross-sectional view of another embodiment of the deliverydevice;

FIG. 6B is a cross-sectional view of an alternative embodiment of thedevice of FIG. 6A;

FIG. 7A is side view of an exemplary embodiment of the delivery devicebeing delivered to a treatment site;

FIG. 7B is a side view of the device of FIG. 7A being positionedadjacent the treatment site; and

FIG. 7C is a side view of the device of FIG. 7A being secured at thetreatment site, and subsequent removal of an insertion tool.

FIG. 8A is a slide showing human articular chondrocytes encapsulated inalginate DME/10% FBS;

FIG. 8B is a slide showing human articular chondrocytes encapsulated inDME/ITS;

FIG. 9A is a slide showing human ligament cells encapsulated in DME/10%FBS;

FIG. 9B is a slide showing human ligament cells encapsulated in DME/ITS;

FIG. 10A is a slide showing human fibroblast cells encapsulated inalginate in DME/10% FBS; and

FIG. 10B is a slide showing human fibroblast cells encapsulated inalginate in DME/IT.

FIG. 11 is a photograph of cultured bovine chondrocytes;

FIG. 12A is a phase contrast photograph of chondrocytes transfected withGreen Fluorescent Protein (GFP);

FIG. 12B is a chondrocyte from FIG. 2A showing expression of GreenFluorescent Protein (GFP);

FIG. 13A is a graph showing expression of EPO from human chondrocytes;

FIG. 13B is a graph showing expression of EPO from bovine chondrocytes;

FIG. 14 is a graph showing expression of EPO mimetibody from humanchondrocytes;

FIG. 15A shows dedifferentiated chondrocytes growing in monolayer;

FIG. 15B is a phase contrast photograph of differentiated chondrocytes;and

FIG. 15C is a photograph of cartilage matrix deposited by thechondrocytes.

DETAILED DESCRIPTION OF THE INVENTION

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the devices and methods disclosed herein. One ormore examples of these embodiments are illustrated in the accompanyingdrawings. Those skilled in the art will understand that the devices andmethods specifically described herein and illustrated in theaccompanying drawings are non-limiting exemplary embodiments and thatthe scope of the present invention is defined solely by the claims. Thefeatures illustrated or described in connection with one exemplaryembodiment may be combined with the features of other embodiments. Suchmodifications and variations are intended to be included within thescope of the present invention.

The practice of the present invention employs, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. (See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.);Fundamental Virology, 2nd Edition, Vol. I & II (B. N. Fields and D. M.Knipe, eds.)).

So that the invention may more readily be understood, certain terms arefirst defined:

The term “chondrocyte” as used herein refers to the art recognized useof the term for a cell type involved in cartilage formation and repair.The chondrocyte functions to produce extracellular matrix ofproteoglycans and collagen. Also included with in the use of the term“chondrocyte” are chondrocyte precursor cells such as chondroblasts, andmesenchymal stem cells.

The term “genetically altered chondrocyte” as used herein refers to achondrocyte cell that has been manipulated using standard molecularbiological techniques, in a way that introduces a heterologous orforeign nucleic acid molecule encoding a therapeutic agent into thecell.

The term “nucleic acid” sequence as used herein refers to a DNA or RNAsequence. The term captures sequences that include any of the known baseanalogues of DNA and RNA such as, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, 2-thiocytosine, and2,6-diaminopurine.

The term “therapeutic agent” as used herein refers to a compound thatproduces a desired therapeutic effect. The therapeutic agent can beselected from the group consisting of a protein (e.g., short termpeptides, bone morphogenic proteins, glycoprotein and lipoprotein), anagonist or an antagonist of an antibody, an antigen, a hormone, ananti-inflammatory agent, an antiviral agent, anti-rejection agents suchas immunosuppressants and anti-cancer drugs, an anti-bacterial agent, agrowth factor, a cytokine, a hormone, an oncogene, a tumor suppressor, atransmembrane receptor, a protein receptor, a serum protein, an adhesionmolecule, a neurotransmitter, a morphogenetic protein, a differentiationfactor, an enzyme, matrix proteins, and an extracellular matrix protein,iRNA, RNA, or fragment and peptides thereof. The therapeutic agentinclude various growth and/or differentiation agents and fragmentsthereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor(HGF), vascular endothelial growth factors (VEGF), fibroblast growthfactors (e.g., bFGF), platelet derived growth factors (PDGF), insulinderived growth factor (e.g., IGF-1, IGF-II) and transforming growthfactors (e.g., TGF-β I-III), parathyroid hormone, parathyroid hormonerelated peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4; BMP-6;BMP-7; BMP-12; BMP-13; BMP-14), sonic hedgehog, growth differentiationfactors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors(e.g., MP52, (aka rhGDF-5)), cartilage-derived morphogenic proteins(CDMP-1; CDMP-2; CDMP-3). Suitable effectors likewise include theagonists and antagonists of the agents described above. The growthfactor can also include combinations of the growth factors describedabove. If other such substances have therapeutic value, it isanticipated that at least some of these substances will have use in thepresent invention, and such substances should be included in the meaningof “therapeutic agent” and “therapeutic agents” unless expressly limitedotherwise. In a preferred embodiment, the therapeutic agent is proteinsuch as the Erythropoietin (EPO) protein. Other examples of suitableproteins include, but are not limited to, insulin protein, pro-insulinprotein, Remicade, bone morphogenetic protein (BMPs), Transforminggrowth factor-beta (TGF-beta), Platelet-derived growth factor (PDGF),cartilage derived morphogenic protein (CDMP), and MP-52.

In another embodiment, the therapeutic agent is an antibody, an antibodyfragment, or a mimetibody. Examples of a useful mimetibody include butare not limited to EPO mimetibody, Remicade mimetibody, BMP mimetibody,cartilage derived morphogenic protein (CDMP) mimetibody and MP-52. In apreferred embodiment, the antibody is the EPO mimetibody.

In yet another embodiment, the therapeutic agent is a growth factor.Preferred growth factors include, but are not limited to, epidermalgrowth factor, bone morphogenetic protein, vascular endothelial-derivedgrowth factor, hepatocyte growth factor, platelet-derived growth factor,hematopoetic growth factors, heparin binding growth factor, peptidegrowth factors, and basic and acidic fibroblast growth factors. In someembodiments it may be desirable to incorporate genes for factors such asnerve growth factor (NGF), TGF-beta superfamily, which includes BMPs,CDMPs, GDFs, (Growth differentiation factors), and MP-52. In yet anotherembodiment, the therapeutic agent is a receptor. Examples of receptorsinclude, but are not limited to, EPO Receptor, B Cell Receptor, FasReceptor, IL-2 Receptor, T Cell Receptor. EGF Receptor, Wnt, InsulinReceptor, TNF Receptor.

The terms “modifies” or “modified” “modifying” or “modification” areused interchangeably herein and refer to the up-regulation ordown-regulation of the therapeutic agent in a cell associated with adisorder, or at a target region. For example, the genetically modifiedchondrocyte can be used to express a therapeutic agent associated with ablood disorder, e.g., the EPO protein, by delivering the geneticallyaltered chondrocyte to the liver or kidney, or any tissue or organ witha vascular supply to allow EPO to reach the target site or region. Oncethe EPO protein is expressed, it can enter the circulatory system andbind to, and alter the function of the EPO receptor (EPOR). This in turnwill cause a change in the environment associated with the EPO receptor,for example by modifying the signal transduction cascade involving theEPO receptor. Thus, the term “modifies” or “modified” also refers to theincrease, decrease, elevation, or depression of processes or signaltransduction cascades involving the therapeutic agent (e.g., EPO), or aprotein associated with the therapeutic agent (e.g., EPOR).

Modification of the cell may occur directly, for example byoverexpressing EPO in a target region. Alternatively, modification of acell may occur indirectly, for example by the overexpressed EPOinteracting with an EPOR that lead to a changes in downstream signaltransduction cascades involving the EPOR. Non-limiting examples ofmodifications include cell proliferation response, cell differentiation,modifications of morphological and functional processes, under- orover-production or expression of a substance or substances by a cell,e.g., a hormone, growth factors, etc, failure of a cell to produce asubstance or substances which it normally produces, production ofsubstances, e.g., neurotransmitters, and/or transmission of electricalimpulses.

These terms are also used to describe the effect of the expressedtherapeutic agent on the cell, the target region, or the environmentsurrounding the cell or target region. The therapeutic agent isexpressed at a level sufficient to produce a desired therapeutic effectby altering the cell, target region, or the environment surrounding thecell or target region. For example, the expression of EPO in a targetregion, (e.g., the liver), will produce elevated levels of EPO thatalter blood hematocrit, and hemoglobin levels.

The phrases “an atypical chondrocyte environment,” a “non-chondrocytetypical environment,” “an environment not usually associated withchondrocytes,” and an ectopic site are used interchangeably herein andrefer to a surrounding in which chondrocytes are not present. Examplesof an environment not usually associated with chondrocytes include thecentral nervous system (CNS). The CNS which includes the brain andspinal cord, is generally considered immunoprivileged. Other examples ofenvironments that are not usually associated with chondrocytes includesolid organs. Examples of solid organs include, but are not limited to,the heart, kidney, liver and pancreas. Yet another example of anenvironment not usually associated with chondrocytes, is thereproductive organs. In males, the reproductive organs not associatedwith chondrocytes are for example, the testis, vas deferens, and thelike. In females, the reproductive organs not associated withchondrocytes are, for example, the uterus, fallopian tubes, ovaries andthe like. Other examples of an environment not associated withchondrocytes include skin, a subcutaneous pouch, intramuscular andintraperitoneal space.

The term “biocompatible substrate” or “biocompatible matrix” are usedinterchangeably herein, and refer to a material into, or onto which acell population (e.g., a chondrocyte population) can be deposited. Thematerial is suitable for implantation in a subject, and does not causetoxic or injurious effects once implanted in the subject.

The term “subject” as used herein refers to any living organism capableof eliciting an immune response. The term subject includes, but is notlimited to, humans, nonhuman primates such as chimpanzees and other apesand monkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

The term “vector” as used herein refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors.

The term “transfection” as used herein refers to the uptake of foreignDNA by a cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. Such techniques can be used tointroduce one or more exogenous DNA moieties, such as a nucleotideintegration vector and other nucleic acid molecules, into suitable hostcells. The term captures chemical, electrical, and viral-mediatedtransfection procedures.

Various aspects of the invention are described in more detail in thefollowing subsections:

I. Culturing and Genetically Altering A Chondrocyte Cell Population

One aspect of the invention pertains to culturing and geneticallyaltering chondrocytes such that the genetically altered chondrocytesexpress a therapeutic agent. In one embodiment, the genetically alteredchondrocytes are used to produce a therapeutic agent in an environmentthat is not typically associated with chondrocytes, such as the brain,or solid organs (e.g., heart, kidney, liver). In another embodiment, thegenetically altered chondrocytes are used to express a therapeutic agentin an environment associated with chondrocytes, such as the cartilage,tendons, joints, and bones. In such instances, the genetically alteredchondrocytes are used exclusively for manufacturing and expressing thetherapeutic agent, and not as components of a tissue engineeredconstruct used to repair or replace a damaged cartilage, tendon, joint,or bone.

There are a number of reasons for using genetically alteredchondrocytes. To begin with, chondrocytes have the ability to survive invery harsh conditions such as environments with a low pH, or low oxygentension. Thus, the genetically altered chondrocyte population has apost-implantation survival advantage, especially when introduced intodiseased or injured tissues such as stroke-damaged tissue.

Furthermore, the genetically altered chondrocytes require no vascularsupport for survival. This is an important since chondrocytes have abetter chance at surviving encapsulation procedures, such as within agel matrix, as well as implantation procedures. In the past, the sizeand geometry of cell containing capsules have been limited because mostcells depend upon close proximity to vascularity for survival. That is,cells encapsulated in too large a format or in large capsules would havecompromised viability because the cells in the center of the implantwould not have adequate gas and nutrient exchange.

In addition, chondrocytes have a uniform phenotype. The tissue fromwhich the cells are isolated is mostly composed of chondrocytes, and itis free of vascular endothelial cells, stromal cells or any other cellthat forms the cartilage tissue. The physical appearance of thecartilage tissue is also very distinct from the surrounding tissue,making dissection very easy. These characteristics allow for easyisolation of pure population normal chondrocytes. Once the isolatedchondrocytes are placed in tissue culture, they behave similarly tofibroblastic cell lines. The fibroblast-like growth is reversible andthe cells can be easily induced to redifferentiate into chondrocyteswhen placed in anchorage independent conditions such as in agarose oralginate gel suspension. Therefore, immortalized transformed cell lines,which are known to be highly metabolic, with a resultant tendencytowards cancerous mutation, are not necessary.

Chondrocytes typically secrete an extensive extracellular matrix whenplaced in anchorage independent conditions This matrix is thought toprotect the cells from direct cell-cell contact from the blood supply,thereby delaying or preventing immune rejection. This could provide yetanother survival advantage and enable an allogenic or xenogenic implantapproach. Allogenic and xenogenic cells could also have a more simpleregulatory pathway since they could be seen as temporary, and thereforecloser in concept to a device than a permanent tissue transplant.

Furthermore, when the chondrocytes are placed in suspension duringanchorage independent culture, they do not need to form an organizedtissue to perform because the secretion of extracellular matrix, whichprotects against rejection, occurs at the cellular level. Large scalepreparation of chondrocyte-containing gels could be performed, and thedose of the therapeutic could be adjusted by varying the amount or sizeof gel to be implanted without affecting the overall behavior of theimplant. In other words, a small and large implant would differ only inthe number of encapsulated cells, without affecting the biologicalfunctioning of the cells.

The chondrocytes can be isolated from a variety of sources. For example,embryonic chondrocytes can be isolated from sterna (Leboy et al., (1989)J. Biol. Chem., 264:17281-17286; Sullivan et al., (1994) J. Biol. Chem.,269:22500-22506; and Bohme et al., (1995) Exp. Cell Res., 216:191-198),and vertebra (Lian et al., (1993) J. Cellular Biochem., 52:206-219),limb bud mesenchymal cells in micromass cultures (Roark et al., (1994)Develop. Dynam., 200:103-116; and Downie et al., (1994) Dev. Biol.,162:195), growth plate chondrocytes in monolayer (Rosselot et al.,(1994) J. Bone Miner. Res., 9:431-439; Gelb et al., (1990)Endocrinology, 127:1941-1947; and Crabb et al. (1990) J. Bone MineralRes., 5:1105-1112), or pellet cultures (Kato et al., (1988) Proc. Nat.Acad. Sci., 85:9552-9556) have been used to characterize chondrocyteresponses to exogenous factors, many of which function in an autocrinemanner.

Chondrocytes can be obtained by a biopsy of cartilaginous tissue from anarea treated with local anaesthetic with a small amount of lidocaineinjected subcutaneously. The chondrocyte cells from the biopsied tissuecan be expanded in culture. The biopsy can be obtained using a biopsyneedle, a rapid action needle which makes the procedure quick andsimple. In a preferred embodiment, chondrocytes are isolated from donortissue and large cell banks are carefully characterized and optimized toprovide a uniform product.

Chondrocyte cells obtained by biopsy can be cultured and passaged asnecessary. For example, articular cartilage can be aseptically obtainedfrom calf knee joints. Cartilage can be shaved from the end of the bonesusing a scalpel. The tissue can be finely minced in saline solution andsubmitted to collagenase and trypsin digestion for several hours at 37°C. until the tissue has completely digested and single cells aregenerated. The chondrocyte cells isolated from the cartilage tissue canthen be washed from enzyme with DMEM containing 10% fetal bovine serum,and seeded onto tissue culture plastic dishes using about 5,000 cells toabout 10,000 cells per square centimeter. The chondrocyte cells can becultured in DMEM and 10% fetal calf serum with L-glutamine (292 μg/cc),penicillin (100 U/cc), streptomycin (100 μg/cc) at 37° C. in 5% CO₂ withan atmosphere having 100% humidity and expanded in number (See FIG. 1).The chondrocyte cells can then be removed from the tissue culture vesselusing trypsin/EDTA and passaged into larger tissue culture vessels orencapsulated in gel suspension. Expanded chondrocyte cells can also befrozen according to standard procedures until needed.

Other sources from which chondrocytes can be derived includeadipose-derived cells, mesenchymal stem cells, fetal stem cells,marrow-derived stem cells, and other pluripotent stem cells. Thechondrocytes can be autogeneic, isogeneic (e.g., from an identicaltwin), allogeneic, or xenogeneic.

A number of studies have been conducted on the isolated chondrocytes,from which has emerged the critical role for a number of growth factors,including basic fibroblast growth factor (bFGF), transforming growthfactor beta (TGFβ), insulin-like growth factor-1 (IGF-1), andparathyroid hormone (PTH), which regulate chondrocyte proliferation anddifferentiation. The expression of these factors and their associatedreceptors are maturation dependent and exquisitely regulated (Bohme, etal., (1992) Prog. Growth Factor Res. 4:45-68). Other studies have shownthat vitamins A, C, and D are also required for chondrocyte maturation(Leboy et al., (1994) Microscopy Res. and Technique 28:483-491; Iwamotoet al., (1993) Exp. Cell Res., 207:413-420; Iwamoto et al., (1993) Exp.Cell Res. 205:213-224; Pacifici et al., (1991) Exp. Cell Res. 195:38-46;Shapiro et al., (1994) J. Bone Min. Res. 9:1229-1237; Corvol et al.(1980) FEBS Lett. 116:273-276; Gerstenfeld et al., (1990) Conn. Tiss.Res. 24:29-39; Schwartz et al., (1989) J. Bone Miner. Res. 4:199-207;and Suda, (1985) Calcif Tissue Int., 37:82-90).

The chondrocyte cells may be engineered to incorporate a nucleic acidencoding a therapeutic agent such as proteins, antibodies, regulatorsand cytokines. Other molecules, genes, or nucleic acids that influencecell growth, matrix production, or other cellular functions such as cellcycle may also be used. Nucleic acids may be DNA, RNA, or othernucleotide polymers. Such polymers may include natural nucleosides,nucleoside analogs, chemically modified bases, biologically modifiedbases, intercalated bases, modified sugars, or modified phosphategroups.

In one embodiment, the nucleic acid can be introduced into thechondrocytes as naked DNA, nanocapsules, microspheres, beads, andlipid-based systems such as liposomes, or carriers such as keyholelimpet hemocyanin (KLH) and human serum albumin (See e.g., U.S. Pat. No.6,303,379, to Selden, incorporated herein by reference).

In another embodiment, the chondrocytes may be transfected using vectorsengineered to carry a nucleic acid that encodes the therapeutic agent.The nucleic acid sequences can be cloned into the vector using standardcloning procedures known in the art, as described by Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, ColdSprings Harbor, N.Y. (1982), which is hereby incorporated by reference.Suitable vectors include, but are not limited to plasmid vectors such aspRO-EX (Gibco/BRL), pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK⁺ orKS⁺ (See Stratagene Cloning Systems Catalog from Stratagene, La Jolla,Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX,pET series (See Studier et al., Use of T7 RNA Polymerase to DirectExpression of Cloned Genes, Gene Expression Technology vol. 185 (1990),which is hereby incorporated by reference) and any derivatives thereof.In a preferred embodiment, the plasmid vector is pcDNA3.1. In anotherpreferred embodiment, the plasmid vector is pCEP4.

The gene encoding the therapeutic agent (e.g., EPO) can be operablylinked to other elements regulating expression of the gene, including,but not limited to, a promoter, an enhancer sequence, repressorsequence, TATA box, transcription stop sequence, ribosomal binding site,post-transcriptional regulatory element, etc. One of skill in the artwould appreciate the variety of elements that may be used in combinationwith the therapeutic gene.

Promoters vary in their “strength” (i.e., their ability to promotetranscription), and can be constitutive. For the purposes of expressinga therapeutic gene, it is desirable to use strong promoters in order toobtain a high level of transcription and expression of the therapeuticgene. In one embodiment, the promoter is a constitutive promoter thatincludes, but is not limited to, CMV, RSV promoters, and the like. Otherpromoters include, but are not limited to, LTR or SV40 promoter, the E.coli. lac or trp, T3 and T7 promoters, HSV thymidine kinase, as well asother promoters known to control expression of genes in prokaryotic oreukaryotic cells. In another embodiment, the promoter is specific for aparticular cell type. For example, a promoter is functional in thecentral nervous system (e.g., glial specific promoters, neuron elonasepromoter, and the like); functional in the organs such as the liver(e.g., albumin and alpha1 antitrypsin promoters, and the like, that areactive in hepatocytes); functional in the pancreas (e.g., insulinpromoter, pancreatic amylase promoter and the like); or functional inthe heart (e.g., ventricular myocyte-specific promoter, alpha-MyHC andbeta-MyHC promoter). One skilled in the art will appreciate that thevectors can be readily engineered to include any desired regulatoryelements that are required for the specific transcription of thetherapeutic gene using standard molecular biology techniques.

Also within the scope of the invention are biologically active fragmentsof the therapeutic agent that produce a desired effect. For example, thecomplete gene for EPO can be transfected into a chondrocyte.Alternatively, active fragments of the gene can be used. Thus, thetransfected gene may code for the entire therapeutic agent, or for abiologically active peptide. Also within the scope of the invention arehomologous proteins that are at least 50, 75, or 90% homologous to thetherapeutic agent. Alternatively, a DNA sequence representing afunctional peptide agonist could also be used.

A variety of gene transfection techniques are known in the art. Thenucleic acid sequence can be introduced into cells via transformation,particularly transduction, conjugation, mobilization, orelectroporation. For example, viral vectors such as adenovirus arecommonly used to insert DNA into a variety of cells. Other transfectionmethods include electroporation, calcium-phosphate methods, and lipidbased methods. (See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., 1989; Miller and Calos, eds., Gene TransferVectors for Mammalian Cells, 1987; Ausubel et al., eds., CurrentProtocols in Molecular Biology, 1987; each of which is incorporatedherein by reference). Both stable transfection and transient expressiontechniques may be employed, depending on how long the therapeutic geneshould be expressed.

Those chondrocytes that have been genetically altered to carry theplasmid encoding the therapeutic agent, can be selected by any methodavailable to the skilled artisan. In a preferred embodiment, thegenetically altered chondrocytes are selected using an antibioticmarker, such as neomycin. This allows for the selection of only thosechondrocytes that have been genetically altered. These isolatedchondrocytes can then be amplified to provide a population of cells thatcarries the plasmid encoding the therapeutic agent. These alteredchondrocytes retain the phenotype of a typical chondrocytes, butprimarily express the therapeutic agent. These genetically alteredchondrocyte population does not become a structural part of the cell,target region or the environment surrounding the cell or target region.The genetically altered chondrocytes function only to express thetherapeutic agent.

II. Environments and Therapeutic Agents

The invention pertains to using genetically engineered chondrocytes asvehicles to deliver a therapeutic agent to an environment. In oneembodiment, the environment can be one that is not typically associatedwith chondrocytes, such as the central nervous system, or solid organssuch as the heart, liver or kidney. In another embodiment, theenvironment is one associated with chondrocytes, however, thechondrocytes still have the chondrocyte phenotype and they are usedexpressly to produce the therapeutic agent. The chondrocytes do notperform the function of cartilage tissue (enabling friction-freearticulation), and thus they are not used for tissue repair orconstruction with a tissue engineered construct. In one embodiment, thegenetically altered chondrocytes are engineered to express onetherapeutic agent, (e.g., EPO). In another embodiment, the geneticallyaltered chondrocytes are engineered more than one therapeutic agents.,for example, two therapeutic agents, three therapeutic agents, or morethan three therapeutic agents.

The genetically altered chondrocytes can be delivered to a cell ortarget region using known procedures for delivering vectors. Forexample, the genetically altered chondrocytes can be mixed with a liquidgel and injected to a target region using a surgical syringe. The liquidgel can solidify in situ at the target region. Alternatively, achondrocyte-gel matrix solid can be surgically implanted into a targetregion. The gel matrix solid can be in the form of a thin sheet that canbe rolled or folded, and inserted into the target region using asurgical instrument, and allowed to unfold at the target region (e.g.,interocular lens). The genetically altered chondrocytes alone (i.e.,without being mixed with a substrate), can be injected to a targetregion.

Examples of environments not typically associated with chondrocytesinclude, but are not limited to the following:

(i) Central Nervous System

The genetically altered chondrocytes of the invention can be used toameliorate or modify the function of cells associated with aneurodegenerative or neurological disorder involving the central nervoussystem. Regions of the central nervous system that can be targeted bythe genetically altered chondrocytes of the invention include the cellsin the brain and the spinal cord. Examples of therapeutic agents thatcan be expressed by genetically altered chondrocytes in the centralnervous system include, but are not limited to, receptors (e.g.N-methyl-D-aspartate (NMDA) receptor, GluR receptor (e.g., GluR4,GluR6)); neurotransmitters (e.g., dopamine, acetylecholine, andnorepharine); transporters (e.g., glutamate transporters such asexcitatory amino acid transporters (EAAT)); growth factors (e.g.,neurotrophic factor (GDNF), ciliary derived neuronotrophic factor(CNTF), brain derived neuronotrophic factor (BDNF), neuronotrophin-3(NT3), epidermal growth factor (EGF), fibroblast growth factor (FGF),transforming growth factor-a (TGF-a), transforming growth factor-b(TGF-b), platelet derived growth factor (PDGF)).

a) Suitable Models of Neurodegenerative Diseases

(i) Huntington's Disease

The genetically altered chondrocytes of the invention can be used toameliorate or modify neurodegeneration in a subject withneurodegenerative diseases such as Huntington's disease. Models ofHuntington's disease have been developed in several different animals,for example in the rat (Isacson et al. (1985) Neuroscience 16:799-817),monkey (Kanazawa, et al. (1986) Neurosci. Lett. 71:241-246), and baboon(Hantraye. et al. (1992) Proc. Natl. Acad. Sci. USA 89:4187-4191;Hantraye, et al. (1990) Exp. Neurol. 108:91-014; Isacson, et al. (1989)Exp. Brain Res. 75(1):213-220). These models of Huntington's diseasehave been described as providing effective therapeutic models that arepredictive of therapeutic efficacy in humans.

Neurodegeneration in Huntington's disease typically involvesdegeneration in one or both nuclei forming the striatum or corpusstratium, the caudate nucleus and putamen. Administration of thegenetically altered chondrocytes that express a therapeutic agent tothese regions may modify the function of these regions. The geneticallyaltered chondrocytes may be delivered to these regions using a liquidpolymer gel comprising the genetically altered chondrocytes, anddelivered using standard stereotactic delivery methods to the specificregions of the brain. The therapeutic effects of expressing thetherapeutic agent using genetically altered chondrocytes, can bedetermined by morphological and immunohistochemical studies. Behavioraltests can also be performed using standard techniques, such as the mazetest.

(ii) Parkinson's Disease

Parkinson's disease in humans primarily affects subcortical structures,especially the substantia nigra and loercus caeruleus. It ischaracterized by the loss of dopamine neurons in the substantia nigra,which have the basal ganglia as their major target organ. Several animalmodels of Parkinson's disease have been generated in which effectivetherapies are indicative of therapeutic efficacy in humans. These animalmodels include three rat models (the rats having lesions in substantianigral dopaminergic cells caused by treatment with 6-hydroxydopamine,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or surgicaltransection of the nigral striatal pathway) (See, e.g. Björklund et al.(1982) Nature 298:652-654), a rhesus monkey model (the monkeys havinglesions in substantia nigral dopaminergic cells caused by treatment withMPTP) (See, e.g., Smith, et al. (1993) Neuroscience 52):7-16; Bakay etal. (1985) Appl. Neurophysiol. 48:358-361; Zamir. et al. (1984) BrainRes. 322:356-360), and a sheep model (the sheep having lesions insubstantia nigral dopaminergic cells caused by treatment with MPTP)(Baskin, et al. (1994) Life Sci. 54:471-479). In one embodiment, thegenetically altered chondrocytes can be modified to express atherapeutic agent (e.g., GABA) that ameliorates, of modifies thefunction of cells associated with Parkinson's disease. The therapeuticeffect can be determined as described above for Huntington's disease.

b) Autoimmune Disorders

(i) Diabetes

The genetically altered chondrocytes of the invention can be used toameliorate or modify autoimmune diseases such as diabetes. A summary ofinsulin-dependent diabetes mellitus and its animal models is describedby Wong et al. (1999) Curr Opin Immunol 11:643-647. A few autoantigenshave been associated with Type I diabetes mellitus, for example, insulin(Palmer et al. (1983) Science 222:1337-1339), glutamic aciddecarboxylase (GAD) (Baekkeskov et al. (1990) Nature 347:151-156) andcarboxypeptidase H (Castano. et al. (1991) J. Clin Endocr. Metab,73:1197-1201), and the glycolipids GT3 (Gillard, et al. (1989) JournalImmunol. Methods 142:3826-3832) and GM2-1 (Dotta, et al. (1992)Endocrinology 130:37-42) and PM-1 (U.S. Pat. No. 5,908,627). Thesemodels can be used to investigate the effect of genetically alteredchondrocytes that express a therapeutic agent (e.g., insulin) ondiabetes in the animal.

(ii) Rheumatoid Arthritis

The genetically modified chondrocytes of the invention can also beimplanted in a patient to treat a disease such as Rheumatoid arthritis.In this embodiment, chondrocytes are implanted either in or close to thejoint capsule for the treatment of specific joint that is affected byRheumatoid arthritis, or in an ectopic site for a systemic delivery toall joints of an anti-inflammatory therapeutic agent. Examples of usefulanti-inflammatory therapeutic agents that can be expressed by thegenetically modified chondrocytes to treat Rheumatoid arthritis include,but are not limited to TNF-alpha antagonist and Remicade.

(c) Reproduction

Substances involved in reproduction can also be modified using thegenetically altered chondrocytes of the invention. Suitable animalmodels for reproduction are Sprague-Dawley rats, which are readilyavailable. For example, modifying the function of luteinizinghormone-releasing hormone (LHRH), a hormone regulated by thehypothalamus and involved in the stimulation of ovulation and uterinegrowth (Fueshko et al. (1994) Dev Biol 166:331-348; Hahn et al. (1984)Endocr Res, 10:123-138). Luteinizing hormone-releasing hormone alsoplays a role in male sterility by inhibiting the action of luteinizinghormone-releasing hormone with a synthetic decapeptide (Carelli (1982)Proc Natl. Acad. Sci. USA 79:5392-5395).

Alternatively, the genetically altered chondrocytes can be used todeliver estrogen for post-menopausal hormone therapy in women. Thesegenetically altered chondrocytes can be delivered to the uterus,fallopian tubes, ovaries and the like reproductive organs. Further,since diffusible factors such as hormones, need not be delivered to aspecific target site, unless there is a systemic toxic or unwanted sideeffect, alternative delivery options include local delivery accomplishedby injection through a syringe, surgical implantation, or endoscopicdelivery through a trochar. The genetically altered chondrocytes mayalso be used for sterilization purposes by delivering a sterilizationagent that causes sterility in males. The genetically alteredchondrocytes that express the sterilization agent can be delivered, forexample to the testis. Other male reproductive organs that can betargeted by the genetically altered chondrocytes include, but are notlimited to, testis, urethra, and ureter.

(d) Blood Related Disorders

The genetically altered chondrocytes can be used to express therapeuticagents such as erythropoietin (EPO). EPO is a glycoprotein hormoneproduced primarily by cells of the peritubular capillary endothelium ofthe kidney, and is responsible for the regulation of red blood cellproduction. Secondary amounts of the hormone are synthesized in liverhepatocytes of healthy adults. In premature as well as full terminfants, the liver is the primary site of EPO production. The kidneybecomes the primary site of EPO synthesis shortly after birth. EPOproduction is stimulated by reduced oxygen content in the renal arterialcirculation. Circulating EPO binds to EPO receptors on the surface oferythroid progenitors resulting in replication and maturation tofunctional erythrocytes by an incompletely understood mechanism.

Clinical conditions that give rise to tissue hypoxia including anemia,lung disease, or cyanotic heart disease, lead to increased levels ofserum EPO. Low EPO levels are observed in patients with anemia, patientswith cancer, as well as those with rheumatoid arthritis, HIV infection,ulcerative colitis, and sickle cell anemia. Suppression of EPO synthesisby inflammatory cytokines (e.g., IL-1, TNF-alpha) is believed to occurin certain chronic diseases or cancer.

In contrast, elevation of EPO levels can occur in association with renaldiseases such as hydronephrosis or cysts, or certain tumors, resultingin erthrocytosis. Examples include renal cell carcinoma (hypernephroma),hepatocellular carcinoma, and adrenal gland tumors. Certain bone marrowdisorders, such as myelodysplastic syndrome and aplastic anemia, mayalso be associated with high serum levels of EPO. In the setting of bonemarrow disease, high serum EPO levels are presumably due to thereduction in the number of EPO receptor bearing cells, thereby allowingserum levels to rise. Thus, genetically modified chondrocytes can alsobe used to express EPO antibody to interact with the elevated EPO toreduce the level.

Suitable in an animal model of anemia that can be used to test thegenetically modified chondrocytes are available (See e.g., Hamamori etal., (1995), J. Clinical Investigation, 95:1808-1813, Osborne et al.,(1995) Proc. Natl. Acad. Sci., USA, 92:8055-8058). The geneticallymodified chondrocytes expressing EPO can be implanted into the anemicanimal and determining the level of EPO and the hematocrit of thetreated animal can be measured over time.

(e) Allergies

The genetically modified chondrocytes of the invention can also beimplanted in a patient to treat allergies by effecting the in vivoproduction of the allergens, or their antibodies through the geneticallymodified chondrocytes. Through controlled expression of the allergens,or their antibodies, the patient gradually develops IgG antibodies thatblock the IgE antibodies which result in long term relief fromallergies.

(f) Pain Management

Pain management can be effected through the genetically modifiedchondrocytes as well by modifying the chondrocytes to express opiates orendorphins The genetically modified chondrocytes that express such painmediating substances would be implanted in tissue such as the brain. Inaddition, the chondrocytes can be modified to express nociceptive painrelievers, and the modified chondrocytes could be implanted locally, atthe site of the injury or disease causing pain.

(g) Cancer Treatment

Cancer is a disease that is characterized by uncontrolled growth ofabnormal or cancerous cells, in most instances as a result of an alteredgenome in a single abnormal cell. The alteration in the genome is causedby a mutation in one or more genes wherein the probability of theoccurrence of a mutation is increased by a variety of factors including(i) ionizing radiation, (ii) exposure to chemical substances known ascarcinogens, (iii) some viruses, (iv) physical irritation, and (v)hereditary predisposition.

The genetically modified chondrocytes can be used to deliver therapeuticagents that enhance a subject's immune response to invading metastasesor to either directly or indirectly suppress cancerous cell growth. Suchtherapeutic agents include, but are not limited to, various cytokinessuch as interleukin-2 (IL-2), granulocyte-macrophage colony stimulatingfactor (GM-CSF), interleukin-12 (IL-12) and interferon-gamma(IFN-gamma), anti-angiogenic molecules and tumor associated antigens(Anderson et al., (1990) Cancer Res., 50: 1853, Stoklosa, et al., (1998)Ann Oncol., 9:63, Leibson, et al., (1984) Nature, 309:799, Book, et al.,(1998) Semin. Oncol. 25:381, Salgaller, et al., (1998) J. Surg. Oncol.,68: 122, Griscelli, et al., (1998) Proc. Natl. Acad. Sci. USA, 95:6367).

Suitable models of tumors are available, for example, the mouse tumormodel (See e.g., Hearing et al., (1986) J. Iminunol., 137: 379).Therapeutic doses of anti-tumor molecules can be delivered in a mousetumor model using the genetically altered chondrocytes. In vivo tissueor serum levels of recombinant molecules are measured at varying timepoints following implantation and the effects on tumor development andanimal survival are followed over time. Other art-accepted animal modelsof cancer have been described in Hearing, (1986) J. Immunol., 137:379,Stoklosa et al., (1998) Ann. Oncol., 9:63, Carson et al., (1998) J.Surg. Res., 75:97, Maurer-Gebhard et al., (1998) Cancer Res., 58:2661and Takaori-Kondo et al., (1998) Blood 91:4747).

Therapeutic efficacy of treatment of cancer can be determined bymeasuring changes in clinical parameters such as tumor shrinkage (e.g.at least 5-10% and preferably 25-100%) and/or extended animal survivaltime.

(h) Solid Organ Treatment

The genetically modified chondrocytes can be used to deliver therapeuticagents (directly or in close proximity) to a region in a solid organsuch as the heart, kidney, or liver.

III. Biocompatible Substrates

The genetically altered chondrocytes of the invention can be deliveredto a desired environment by using biocompatible substrates. In oneembodiment of the present invention, the substrate can be formed from abiocompatible polymer. A variety of biocompatible polymers can be usedto make the biocompatible tissue implants or substrates according to thepresent invention. The biocompatible polymers can be synthetic polymers,natural polymers or combinations thereof. As used herein the term“synthetic polymer” refers to polymers that are not found in nature,even if the polymers are made from naturally occurring biomaterials. Theterm “natural polymer” refers to polymers that are naturally occurring.In embodiments where the substrate includes at least one syntheticpolymer, suitable biocompatible synthetic polymers can include polymersselected from the group consisting of aliphatic polyesters, poly(aminoacids), copoly(ether-esters), polyalkylenes oxalates, polyamides,tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters,polyoxaesters, polyamidoesters, polyoxaesters containing amine groups,poly(anhydrides), polyphosphazenes, and blends thereof. Suitablesynthetic polymers for use in the present invention can also includebiosynthetic polymers based on sequences found in collagen, elastin,thrombin, fibronectin, starches, poly(amino acid), poly(propylenefumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose,chitin, chitosan, tropoelastin, hyaluronic acid, ribonucleic acids,deoxyribonucleic acids, polypeptides, proteins, polysaccharides,polynucleotides and combinations thereof.

For the purpose of this invention aliphatic polyesters include, but arenot limited to, homopolymers and copolymers of lactide (which includeslactic acid, D-,L- and meso lactide); glycolide (including glycolicacid); ε-caprolactone; p-dioxanone (1,4-dioxan-2-one); trimethylenecarbonate (1,3-dioxan-2-one); alkyl derivatives of trimethylenecarbonate; δ-valerolactone; β-butyrolactone; γ-butyrolactone;ε-decalactone; hydroxybutyrate; hydroxyvalerate; 1,4-dioxepan-2-one(including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione);1,5-dioxepan-2-one; 6,6-dimethyl-1,4-dioxan-2-one; 2,5-diketomorpholine;pivalolactone; α,α diethylpropiolactone; ethylene carbonate; ethyleneoxalate; 3-methyl-1,4-dioxane-2,5-dione;3,3-diethyl-1,4-dioxan-2,5-dione; 6,6-dimethyl-dioxepan-2-one;6,8-dioxabicycloctane-7-one and polymer blends thereof. Aliphaticpolyesters used in the present invention can be homopolymers orcopolymers (random, block, segmented, tapered blocks, graft, triblock,etc.) having a linear, branched or star structure.Poly(iminocarbonates), for the purpose of this invention, are understoodto include those polymers as described by Kemnitzer and Kohn, in theHandbook of Biodegradable Polymers, edited by Domb, et. al., HardwoodAcademic Press, pp. 251-272 (1997). Copoly(ether-esters), for thepurpose of this invention, are understood to include thosecopolyester-ethers as described in the Journal of Biomaterials Research,Vol. 22, pages 993-1009, 1988 by Cohn and Younes, and in PolymerPreprints (ACS Division of Polymer Chemistry), Vol. 30(1), page 498,1989 by Cohn (e.g., PEO/PLA). Polyalkylene oxalates, for the purpose ofthis invention, include those described in U.S. Pat. Nos. 4,208,511;4,141,087; 4,130,639; 4,140,678; 4,105,034; and 4,205,399.Polyphosphazenes, co-, ter- and higher order mixed monomer basedpolymers made from L-lactide, D,L-lactide, lactic acid, glycolide,glycolic acid, para-dioxanone, trimethylene carbonate and, -caprolactonesuch as are described by Allcock in The Encyclopedia of Polymer Science,Vol. 13, pages 31-41, Wiley Intersciences, John Wiley & Sons, 1988 andby Vandorpe, et al in the Handbook of Biodegradable Polymers, edited byDomb, et al., Hardwood Academic Press, pp. 161-182 (1997).Polyanhydrides include those derived from diacids of the formHOOC—C₆H₄—O—(CH₂)_(m)—O—C₆H₄—COON, where “m” is an integer in the rangeof from 2 to 8, and copolymers thereof with aliphatic alpha-omegadiacids of up to 12 carbons. Polyoxaesters, polyoxaamides andpolyoxaesters containing amines and/or amido groups are described in oneor more of the following U.S. Pat. Nos. 5,464,929; 5,595,751; 5,597,579;5,607,687; 5,618,552; 5,620,698; 5,645,850; 5,648,088; 5,698,213;5,700,583; and 5,859,150. Polyorthoesters such as those described byHeller in Handbook of Biodegradable Polymers, edited by Domb, et al.,Hardwood Academic Press, pp. 99-118 (1997).

As used herein, the term “glycolide” is understood to includepolyglycolic acid. Further, the term “lactide” is understood to includeL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers.

Elastomeric copolymers are also particularly useful in the presentinvention. Suitable elastomeric polymers include those with an inherentviscosity in the range of about 1.2 dL/g to 4 dL/g, more preferablyabout 1.2 dL/g to 2 dL/g and most preferably about 1.4 dL/g to 2 dL/g asdetermined at 25° C. in a 0.1 gram per deciliter (g/dL) solution ofpolymer in hexafluoroisopropanol (HFIP). Further, suitable elastomersexhibit a high percent elongation and a low modulus, while possessinggood tensile strength and good recovery characteristics. In thepreferred embodiments of this invention, the elastomer exhibits apercent elongation greater than about 200 percent and preferably greaterthan about 500 percent. In addition to these elongation and modulusproperties, suitable elastomers should also have a tensile strengthgreater than about 500 psi, preferably greater than about 1,000 psi, anda tear strength of greater than about 50 lbs/inch, preferably greaterthan about 80 lbs/inch.

Exemplary biocompatible elastomers that can be used in the presentinvention include, but are not limited to, elastomeric copolymers ofε-caprolactone and glycolide (including polyglycolic acid) with a moleratio of ε-caprolactone to glycolide of from about 35:65 to about 65:35,more preferably from 45:55 to 35:65; elastomeric copolymers ofε-caprolactone and lactide (including L-lactide, D-lactide, blendsthereof, and lactic acid polymers and copolymers) where the mole ratioof ε-caprolactone to lactide is from about 35:65 to about 65:35 and morepreferably from 45:55 to 30:70 or from about 95:5 to about 85:15;elastomeric copolymers of p-dioxanone (1,4-dioxan-2-one) and lactide(including L-lactide, D-lactide, blends thereof, and lactic acidpolymers and copolymers) where the mole ratio of p-dioxanone to lactideis from about 40:60 to about 60:40; elastomeric copolymers ofε-caprolactone and p-dioxanone where the mole ratio of ε-caprolactone top-dioxanone is from about from 30:70 to about 70:30; elastomericcopolymers of p-dioxanone and trimethylene carbonate where the moleratio of p-dioxanone to trimethylene carbonate is from about 30:70 toabout 70:30; elastomeric copolymers of trimethylene carbonate andglycolide (including polyglycolic acid) where the mole ratio oftrimethylene carbonate to glycolide is from about 30:70 to about 70:30;elastomeric copolymers of trimethylene carbonate and lactide (includingL-lactide, D-lactide, blends thereof, and lactic acid polymers andcopolymers) where the mole ratio of trimethylene carbonate to lactide isfrom about 30:70 to about 70:30; and blends thereof. Examples ofsuitable biocompatible elastomers are described in U.S. Pat. No.5,468,253.

In one embodiment, the elastomer is a copolymer of 35:65 ε-caprolactoneand glycolide, formed in a dioxane solvent and including a polydioxanonemesh. In another embodiment, the elastomer is a copolymer of 40:60ε-caprolactone and lactide with a polydioxanone mesh. In yet anotherembodiment, the elastomer is a 50:50 blend of a 35:65 copolymer ofε-caprolactone and glycolide and 40:60 copolymer of ε-caprolactone andlactide. The polydioxanone mesh may be in the form of a one layer thicktwo-dimensional mesh or a multi-layer thick three-dimensional mesh.

In another embodiment, the substrate can be in the form of an injectablegel matrix. The gel matrix can be a biological or synthetic hydrogel,including alginate, cross-linked alginate, hyaluronic acid, collagengel, fibrin glue, fibrin clot, poly(N-isopropylacrylamide), agarose,chitin, chitosan, cellulose, polysaccharides, poly(oxyalkylene), acopolymer of poly(ethylene oxide)-poly(propylene oxide), poly(vinylalcohol), polyacrylate, platelet rich plasma (PRP) clot, platelet poorplasma (PPP) clot, Matrigel, or blends thereof.

The gel matrix is selected to have properties that allow the therapeuticagent to be expressed within the gel matrix, as well as allowing theexpressed therapeutic agent to diffuse out of the gel matrix into thesurrounding environment.

Examples of gel matrices that may be used include, but are not limitedto, collagen gel, fibrin glue, polyglycolic acid, polylactic acid,polyethylene oxide gel, alginate or calcium alginate gel,poly-(2-hydroxyethyl methacrylate) (i.e., a hydrogel), polyorthoester,hyaluronic acid, polyanhydride, chitosan, gelatin, agarose, and otherbioresorbable and biocompatible materials such as those described in EP0705878 A2. To promote chondrocyte proliferation and function, thebiological gel can additionally contain appropriate nutrients (e.g.,serum, salts such as calcium chloride, ascorbic acid, and amino acids)and growth factors (e.g., somatomedin, basic fibroblast growth factor,transforming growth factor-β, cartilage growth factor, bone-derivedgrowth factor, or a combination thereof).

Polysaccharides are a class of macromolecules of the general formula(CH₂O)n which are also useful as the hydrogel substrate in the presentinvention. Polysaccharides include several naturally occurringcompounds, e.g., agarose, alginate and chitosan.

Agarose is a clear, thermoreversible hydrogel made of polysaccharides,mainly the alternating copolymers of 1,4 linked and3,6-anhydro-α-L-galactose and 1,3 linked β-D-galactose. Two commerciallyavailable agaroses are SeaPrep™ and SeaPlaque™ agarose (FMC Corp.Rockland, Me.). The thermoreversible properties of agarose gels make itpossible for agarose to be a liquid at room temperature allowing foreasy mixing of cell-gel solution and then cooling to 4° C. causesgelation and entrapment of cells. This is a comparatively benignprocess, free of chemicals toxic to the cells.

The agarose concentration can be about 0.50 to 2% (w/v), most preferablyabout 1.0%. In any event, the concentration of agarose should besufficient to permit chondrocyte encapsulation at a concentration thatvaries in the range of about 10⁴ cells/ml to 10⁷ cells/ml, and morepreferably in the range of about 10⁵ to 10⁶ cells/ml.

Alginate is a carbohydrate polymer isolated from seaweed, which can becrosslinked to form a hydrogel by exposure to a divalent cation such ascalcium, as described, for example in WO 94/25080, the disclosure ofwhich is incorporated herein by reference. The modified alginatesolution is mixed with the cells to be implanted to form a suspension.Then the suspension is injected directly into a patient prior tocrosslinking of the polymer to form the hydrogel containing the cells.The suspension then forms a hydrogel over a short period of time due tothe presence in vivo of physiological concentrations of calcium ions(See e.g., U.S. Pat. No. 4,352,883 to Lim, incorporated herein byreference). It is also feasible to form the hydrogel first prior toinjection into a patient.

In general, the polymers that make up the biological gels are at leastpartially soluble in aqueous solutions, such as water, buffered saltsolutions, or aqueous alcohol solutions. Methods for the synthesis ofthe polymers are known to those skilled in the art (See e.g., ConciseEncyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts,E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980)). Naturallyoccurring and synthetic polymers may be modified using chemicalreactions available in the art and described, for example, in March,“Advanced Organic Chemistry,” 4th Edition, 1992, Wiley-IntersciencePublication, New York.

Water soluble polymers with charged side groups may be crosslinked byreacting the polymer with an aqueous solution containing ions of theopposite charge, either cations if the polymer has acidic side groups oranions if the polymer has basic side groups. Examples of cations forcrosslinking of the polymers with acidic side groups to form a hydrogelare monovalent cations such as sodium, and multivalent cations such ascopper, calcium, aluminum, magnesium, strontium, barium, and tin, anddi-, tri- or tetra-functional organic cations such as alkylammoniumsalts. Aqueous solutions of the salts of these cations are added to thepolymers to form soft, highly swollen hydrogels and membranes. Thehigher the concentration of cation, or the higher the valence, thegreater the degree of cross-linking of the polymer. Additionally, thepolymers may be crosslinked enzymatically, e.g., fibrin with thrombin.

Several physical properties of the biological gels are dependent upongel concentration. Increase in gel concentration may change the gel poreradius, morphology, or its permeability to different molecular weightproteins. Gel pore radius determination can be effected by any suitablemethod, including hydraulic permeability determination using a graduatedwater column, transmission electron microscopy and sieving spheres ofknown radius through different agar gel concentrations (See, e.g.,Griess et al., (1993) Biophysical J., 65:138-48).

A variety of other suitable biological gels are also known. The polymercan be mixed with cells for implantation into the body and can becrosslinked to form a hydrogel matrix containing the cells either beforeor after implantation in the body. A hydrogel is defined as a substanceformed when an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.

One skilled in the art will appreciate that the volume or dimensions(length, width, and thickness) of the biological gel comprising thechondrocytes can be selected based on the region or environment intowhich the biological gel substrate is to be implanted. In oneembodiment, the biological gel has a length (defined by a first andsecond long end) of about 10 cm to about 30 cm. The biological gel canfurther have a length of about 15 cm to about 25 cm. In a preferredembodiment, the biological gel has a length of about 20 cm. In anotherembodiment, the biological gel has a width (defined by a first andsecond short end) of about 0.5 cm to about 4.0 cm. The biological gelcan further have a width of about 1.0 cm to about 3.0 cm. In a preferredembodiment, the biological gel has a biocompatible substrate with widthof about 2.0 cm.

IV. Manufacturing of Therapeutic Agents Using Genetically AlteredChondrocytes

In another aspect, the invention pertains to using the geneticallyaltered chondrocytes for large scale in vitro preparation of therapeuticagents. For example, liter scale production (e.g., several liters) couldbe effected with expansion and maintenance of chondrocytes onmicrocarrier beads. Chondrocyte cells could be genetically manipulatedto express a therapeutic molecule of commercial value, then expanded andseeded onto microcarrier-bead containing bioreactor. Alternatively,chondrocytes could be seeded directly onto microcarrier beads andexpanded directly in bioreactors.

V. Delivery Devices

Various exemplary embodiments of devices and methods adapted to delivertherapeutic agents produced by genetically-altered chondrocytes areprovided herein. More specifically, the devices have an external housingwhich defines an internal cell chamber. The cell chamber is configuredto retain a large-volume of chondrocytes while allowing for the releaseof therapeutic agents produced by these entrapped cells. As will beseen, the cell chamber is sized such that a core of the chamber can be arelatively large distance away from any external nutrient supply therebyutilizing the enhanced viability of chondrocytes to deliver a largevolume of cells, and subsequently a large amount of therapeutic agent tothe treatment site. More specifically, the cell chamber can beconfigured such that at least a portion of the cells entrapped thereincan be positioned at least about 1.5 mm away from any external nutrientsupply. For example, a tubular cell chamber can be sized so as to haveboth a length and a diameter of at least about 3 mm thereby requiringany external nutrients to diffuse about 1.5 mm to reach the core of thechamber. As will be described below, the release rate of the therapeuticagent can be controlled by positioning various exit ports (e.g.,semi-permeable membranes) at various locations relative to the cellchamber. In some embodiments, the entire housing can include such asemi-permeable membrane. In other exemplary embodiments, the housing candefine additional chambers capable of interacting with the cell chamberto modify the efficiency of the chondrocytes and/or the rate of releaseof the therapeutic agents. These components and others are now describedin detail.

FIG. 1A provides an exemplary embodiment of a delivery device 10provided herein. As shown, the device 10 includes a housing 12 having adistal end 14, a proximal end 16, and further defining an internal cellchamber 18 (see FIG. 1C) adapted to retain a plurality ofgenetically-altered chondrocytes. The housing 12 can include a varietyof configurations and/or materials so as to optimize delivery andplacement of the delivery device 10. For example, the embodiments ofFIGS. 1A and 1B provide a substantially tubular housing 12, and theembodiment of FIG. 3 provides a substantially disc-shaped housing 12.Those skilled in the art will appreciate that various suchconfigurations and/or shapes are within the spirit and scope of thepresent invention.

Additionally, the housing 12 can include both rigid and non-rigidmaterials which are selected, sized and positioned to optimize deliveryof the device, placement of the device at the treatment site, and, aswill be explained in further detail below, release rates of thetherapeutic agents produced therein. Referring to FIGS. 1A and 1B, thehousing can include a rigid frame 17 configured to allow the deliverydevice 10 to maintain shape during the therapeutic regimen. Also, asshown in FIG. 1B, the rigid frame 17 can allow for a tapered or pointeddistal end 14′ configured to facilitate insertion and delivery of thedevice 10′. Those skilled in the art will appreciate that the rigidframe 18 can be formed from various biocompatible, rigid materials. Forexample, the materials can include various metals, metal alloys,polymers, combination of polymers, or any combination thereof.

Referring to FIGS. 2A-2C, various embodiments of the delivery device 100can also include a housing 12′ having a non-rigid frame 17′. Use of anon-rigid frame 17′ can provide a housing 12′ capable of changingconfiguration during delivery of the device 100 to the treatment site,and/or allowing the housing 12′ to substantially adopt the shape of thetreatment site upon arrival at the site thereby enhancing delivery ofthe therapeutic agents. As will be apparent to those skilled in the art,the non-rigid frame 17′ of the housing 12′ can be formed from awide-range of biocompatible, non-rigid materials. For example, thenon-rigid materials can include various polymers adapted to prevent thepassage of both chondrocytes and the therapeutic agents producedtherefrom (i.e., a non-permeable membrane).

The delivery device 10 further includes at least one exit port 20incorporated into the housing 12, and in communication with the internalcell chamber 18. As will be discussed, the exit port 20 is configured toretain chondrocytes within an internal cell chamber 18 while allowingfor the release of a therapeutic agent produced via these entrappedcells. The exit port 20 can be any mechanism capable of such selectiverelease of the therapeutic agents. In an exemplary embodiment, the exitport 20 is a semi-permeable membrane. Those skilled in the art willappreciate that any such membrane 20 capable of retaininggenetically-altered chondrocytes while allowing for the release oftherapeutic agent(s) produced by these entrapped cells is within thespirit and scope of the present invention. For example, thesemi-permeable membrane can include a polytetrafluorethylene (“PTFE”)membrane with pores ranging from about 0.1 mm to about 0.7 mm. In oneembodiment, the pore size is about 0.4 mm to about 0.5 mm. In anotherembodiment, the pore size is about 0.2 mm.

Exit ports 20 of various sizes, shapes, and/or numbers can beincorporated into the device so as to increase/decrease the release rateof the therapeutic agents. Referring to FIG. 1A, the device 10 includesa first and a second rectangular-shaped exit port 20 extending along aside of the housing 12. FIGS. 2A-2C can include an exit port 20 beingincorporated anywhere within the non-rigid housing 12′ (indicatedgenerally by an arrow extending towards the housing 12′). For example,the housing 12′ can include non-permeable portions and a select area(e.g., the distal tip of the device 100) having a semi-permeablemembrane 20 which is adapted to retain chondrocytes while releasing thetherapeutic agents produced therein. In other embodiments, the entirehousing 12′ can be formed from the semi-permeable membrane 20.

Looking at other examples, FIG. 3 provides a disc-shaped housing havinga top portion 50, a bottom portion 50′, and a central ring 52 joiningthe top 50 and bottom portions 50′. Similar to the embodiments of FIGS.2A-2C, the entire top 50 and bottom 50′ portions of the device 200 canbe formed of a semi-permeable membrane 20, or each portion 50, 50′ caninclude a semi-permeable membrane 20 incorporated into a non-permeablematerial. In other embodiments, the top portion 50 of the device 200 canbe formed of a semi-permeable membrane 20, and the bottom portion 50′can be formed of a non-permeable membrane. Those skilled in the art willrecognize that any other combination of semi-permeable and non-permeablemembranes are also within the spirit and scope of the present invention.

Referring to the embodiments of FIGS. 4A-6B, the exit port 20 can beincorporated at the distal tip of the device. Such placement of the exitport 20 can allow for the better predictability as to release rateand/or better concentration of the therapeutic agent at the treatmentsite. Also, use of such a single distal exit port 20 can allow for thetherapeutic agent to be delivered directly to a delivery tube 80 forsubsequent transport of the agent to various distant locations (as willbe discussed below). As will be appreciated by those skilled in the art,embodiments employing a distal exit port 20 can also utilize any numberand/or positions of additional exit ports 20 and remain within thespirit and scope of the present invention.

The various housings discussed above each define an internal cellchamber 18 adapted to retain a plurality of genetically-alteredchondrocytes. Furthermore, the cell chambers 18 are further incommunication with at least one exit port 20 thereby allowing for therelease of the therapeutic agents produced via the entrapped cells. Asdiscussed above, the release rate of these therapeutic agents can bemodified by changing the number, size, character, pore size, etc. of theexit port 20 in communication with the cell chamber 18. Similar to theabove-discussion of the housing, the cell chamber 18 can take variousshapes, sizes, and/or configurations. In general, the cell chamber 18corresponds to the shape of the housing 12 thereby resulting in a cellchamber 18 having a configuration which is tubular (FIGS. 1A-1B), discshaped (FIG. 3), etc.

As mentioned above, use of chondrocytes allows for a cell chamber 18capable of retaining a large volume of chondrocytes without beingrequired to minimize the diffusion distance between the core of the cellchamber 18 and an external nutrient supply (i.e., an outer wall of thedevice). More specifically, in an exemplary embodiment, the cell chamber18 can be sized such that at least a portion of the chondrocytesretained with the cell chamber 18 can be positioned at least about 1.5mm from an external source of nutrients. For example, referring to FIGS.1B (showing a length ‘L’ of the internal cell chamber 18) and 1C(showing a diameter ‘D’ of the cell chamber 18), the cell chamber 18 canbe sized such that the length (L) and the diameter (D) of the cellchamber 18 are each at least about 3 mm thereby resulting in at least aportion of the cells being at least about 1.5 mm displaced from anexternal nutrient supply (i.e., the outer housing 12 of the device 10).In other exemplary embodiments, the cell chamber 18 can have a length(L) in the range of from about 100 mm to about 300 mm, and a diameter(D) in the range of from about 3 mm to about 20.0 mm. In otherembodiments, the cell chamber 18 can have a length (L) in the range offrom about 150 mm to about 250 mm. In another embodiment, the cellchamber 18 has a length (L) of about 200 mm and a diameter (D) of about10 mm. In another embodiment, the cell chamber 18 has a length (L) inthe range of from about 15 mm to about 50.0 mm, and a diameter (D) offrom about 10.0 mm to about 30.0 mm.

The delivery device 10 can allow for the introduction of chondrocytes tothe cell chamber 18 in a variety of manners. Generally, as introduced tothe cell chamber 18, the chondrocytes reside in a gel, gel-like matrix,a liquid suspension, dispersed on a substrate, etc. By way ofnon-limiting example, the chondrocytes can reside in the biocompatiblesubstrates discussed above in section III. Those skilled in the art willappreciate that chondrocytes in any such form are within the spirit andscope of the present invention. Several examples of such forms which arecapable of being utilized are disclosed in assignees co-pendingapplications U.S. Published Application No. 2005/0054595 and U.S.Published Application No. 2006/0292131, the entirety of theseapplications being incorporated herein by reference. These applicationsalso detail the preparation of such genetically-altered chondrocytes.Further, the therapeutic agents and examples of potential treatmentsites are detailed below in relation to the methods provided herein.

Referring to FIGS. 1A-1B, the delivery device 10, 10′ can include aremovable cap 22, 22′ which allows for introduction of the chondrocytecells (as indicated in the FIGS. by dots) to the cell chamber 18. Asshown in the embodiment of FIG. 1B, the cap 22′ can include an headhaving a series of flats (e.g., a series of flats forming a hexagonshape) which facilitates grasping of the cap by a tool (not shown).Referring to FIG. 1A, the cap 22 can also include a series of grooves 24adapted to engage a corresponding set of grooves 24′ incorporated intoan inner wall of the housing 12 thereby allowing the cap 22 to engage ordisengage (i.e., screw-on, screw-off) the housing 12. Referring to FIGS.2A-2C, those embodiments of the device 100 having a housing formed of anon-rigid frame 17′ can utilize a compression ring such as a hose clamp23 or a heat shrink band 25 to engage the cap to the housing 12′. Asalso shown in the embodiments of FIGS. 1A-1B and 2A-2C, the cap 22 caninclude additional features such as a radiopaque marker 28 for enhancedvisualization of the delivery device 10 once implanted, and/or a sutureloop 26 adapted to allow the delivery device 10 to be anchored at thetreatment site.

In other embodiments, the delivery device can be adapted to allow forthe introduction of chondrocytes (indicated in the FIGS. by circleswhich are clearly not to scale) into a sealed cell chamber 18 such aswhen the device 10 is already positioned at the treatment site. Forexample, as shown in FIGS. 3 and 6B, the delivery device can include ainduction port 30 adapted to allow for injection of chondrocytes to thecell chamber 18 via a syringe 32. The induction port 30 an be anymechanism adapted to be pierced by the syringe 32 while remainingcapable of maintaining a closed environment. For example, the inductionport 30 can be a rubber septum incorporated into the housing 12. Thoseembodiments of the delivery device 10 utilizing such an induction port30 can be replenished with new chondrocytes without having to remove thedevice 10 from the treatment site thereby allowing for enhancedefficiency.

In other exemplary embodiments, the housing 12 of the device 300 caninclude various additional chamber(s) so as to control the delivery ofthe therapeutic agents and/or to provide auxiliary fluids (e.g., cellnutrients, etc.) to the cell chamber 18. Referring to FIGS. 4A and 4B,the housing 12 can define an expandable chamber 60 separated from thecell chamber 18 via a piston 62. The expandable chamber 60 can include awater-swellable agent such that in response to the introduction of waterinto the expandable chamber 60, the water swellable material expands anddrives the piston in the distal direction (as shown in FIG. 4B). As thepiston 62 moves distally, the volume of the cell chamber 18 decreasesthereby forcing an amount of therapeutic agent out of the distal exitport 20 of the cell chamber 18 (as indicated by a series of arrows).

As will be apparent to those skilled in the art, the water swellablematerial can be any material cable of expanding in response to theintroduction of water. Additionally, as will also be apparent to thoseskilled in the art, the delivery device 300 can be adapted in variousmanners so as to allow for the introduction of water to the expandablechamber 60. For example, as shown in FIGS. 4A and 4B, the expandablechamber 62 can include an osmotic membrane 64 adapted to allow for theintroduction of water into the expandable chamber 60.

In other exemplary embodiments, the delivery device 400 can include anauxiliary fluid chamber 70 adapted to retain various auxiliary fluids,and deliver these auxiliary fluids to the cell chamber 18. As will beapparent to those skilled in the art, the auxiliary fluid can be anyfluid deemed necessary and/or desirable for a given procedure. Forexample, the auxiliary fluid can include various cell nutrients, or theauxiliary fluid can be any agent capable of modifying or enhancingchrondrocyte performance. Referring to FIGS. 5A-5C, such the auxiliaryfluid chamber 70 can be positioned between the cell chamber 18 and theexpandable chamber 60. In such an embodiment, as the expandable chamber60 expands (as discussed above), the piston 62 is driven in the distaldirection. However, in this embodiment, the auxiliary fluid chamber 70can be positioned adjacent the piston 62. As shown, a semi-permeablemembrane 72 can be disposed between the auxiliary fluid chamber 70 andthe cell chamber 18 such that the auxiliary fluid is controllablyintroduced to the population of chondrocytes in response to theexpanding water-swellable compartment. As shown in FIG. 5B, introductionof the auxiliary fluid to the cell chamber 18 can forces the therapeuticagent(s) out of the distal exit port 20.

In an alternative embodiment, an osmotic pump, such as that of the DUROSdevice described in U.S. Pat. Nos. 6,287,295; 6,156,331; 6,395,292;6,261,584; and 6,635,268; the disclosures of which are incorporated byreference, could provide for the flow of therapeutic agent. Optimalcontrol could further be provided for by the HAKIM valve mechanism.

Referring to FIG. 5C, other embodiments of the delivery device 400′ canbe adapted to deliver the therapeutic agent(s) from the cell chamber 18to a distal location (i.e., any location not immediately adjacent theexit port 20). As shown, the distal exit port 20 can be coupled to adelivery tube 80 so as to deliver the therapeutic agent to the distallocation. As will be apparent to those skilled in the art, the deliverytube 80 can have any dimensions and/or any length capable of being influid communication with the exit port 20, and further capable ofdelivering the therapeutic agent to the desired treatment site.Additionally, the delivery tube 80 can be adapted so to be able todeliver the therapeutic agent to various distant locations. For example,in the embodiment of FIGS. 5C and 6A, the delivery tube 80 include afirst branch 82 adapted to deliver a portion of the therapeutic agent toa first distal location, and a second branch 84 adapted to deliveranother portion of the therapeutic agent to a second distal location.Those skilled in the art will appreciate that a delivery tube having oneor any additional number of such branches is within the spirit and scopeof the present invention.

In other embodiments, an external pump 90 and/or valve system 88 can becoupled to the delivery device 500 to provide enhanced control of thedelivery of the therapeutic agent from the device 500. Referring to FIG.6A, an external pump 90 can be coupled to the delivery device 500 via avalve element 88 thereby allowing for continuous pressurized flow of anauxiliary fluid from an external reservoir (not shown). Additionally,introduction of the fluid via the pump 90 can also provide substantiallycontinuous outflow of therapeutic agents from the device 500.Additionally, the pump 90 can be utilized to infuse the cell chamber 18with various toxic agents so as to deactivate the chondrocytes uponcompletion of the desired therapeutic regimen. This configuration issomewhat analogous to the HAKIM shunt for hydrocephalitis, except thatit is functioning in the reverse direction to actively deliver a fluidinto the brain from a reservoir, rather than passively removing a fluidfrom the brain and draining it into the peritoneal cavity. The valvemechanism of the HAKIM shunt, which is described in U.S. Pat. Nos.4,332,255, 4,387,715, 4,551,128, 4,595,390, and 4,615,691, thedisclosures of which are incorporated herein by reference, could be usedto remotely adjust the flow of the therapeutic agent transcutaneously,without the need for additional surgery.

In addition to the embodiments discussed above, a method for deliveringtherapeutic agents to a treatment site is also provided. As described,the device can contain genetically-altered chondrocytes adapted toproduce and release various therapeutic agents. While the chondrocytesare retained within a large-scale cell chamber of the delivery device,various therapeutic agents can be produced within the device via thechondrocytes and delivered to the treatment site via an exit port incommunication with the cell chamber.

The method can include disposing a plurality of chondrocytes into thecell chamber of the delivery device. Similar to the embodimentsdiscussed above, the cell chamber can be sized such that a portion ofthe chondrocytes can be positioned within the cell chamber at a distanceof at least about 1.5 mm from an external source of cell nutrients(e.g., a tubular cell chamber having a length and diameter both of whichare greater than at least about 3 mm). Further, the cell chamber can beadapted so as to retain the chondrocytes while allowing for the releaseof a therapeutic agent(s) produced by the chondrocytes. The methodfurther includes delivering the delivery device to a treatment site, andthereafter, delivering the therapeutic agent from the device to thetreatment site. In other embodiments, the method can further includesuturing the delivery device at the treatment site, and/or injectingadditional chondrocytes into the delivery device.

FIGS. 7A-7C provides a representation of the device 10 being deliveredto the treatment site 96. As shown in FIG. 7A, the device 10 can bemated to an insertion tool 94. As will be apparent to those skilled inthe art, various type of insertion tools 94 can be mated to the device10 in a wide variety of manners thereby allowing the tool 94 toaccurately deliver the device 10 to the treatment site 96. Next, FIG. 7Bshows the device 10 being positioned at the treatment site 96. As shown,the device can pierce the treatment site 96 (e.g., a tissue) therebyallowing the device to be disposed substantially within the site 96. Asshown in FIG. 7C, the device 10 can then be disengaged from theinsertion tool 94, and the tool 94 can be removed from the site 96. Oncedisengaged, the device 10 can be secured to the site 96 via the sutureloop 26, and the device can subsequently deliver various therapeuticagent(s) to the site 96.

Various such therapeutic agents can be produced and released via thesegenetically-modified chondrocytes. In general, the therapeutic agent canbe any compound that produces a desired therapeutic effect. For example,the therapeutic agent can be selected from the group consisting of aprotein, an agonist or an antagonist of an antibody, an antigen, ahormone, an anti-inflammatory agent, an antiviral agent, ananti-bacterial agent, a growth factor, a cytokine, an oncogene, a tumorsuppressor, a transmembrane receptor, a protein receptor, a serumprotein, an adhesion molecule, a neurotransmitter, a morphogeneticprotein, a differentiation factor, an enzyme, a matrix protein, anextracellular matrix protein, iRNA, RNA, or fragments and peptidesthereof. In an exemplary embodiment, the therapeutic agent is a proteinsuch as the Erythropoietin (EPO) protein. Other examples of suitableproteins include, but are not limited to, insulin protein, pro-insulinprotein, Remicade, bone morphogenetic protein (BMPs), Transforminggrowth factor-beta (TGF-beta), Platelet-derived growth factor (PDGF),cartilage derived morphogenic protein (CDMP), and MP-52.

In another embodiment, the therapeutic agent is an antibody, an antibodyfragment, or a mimetibody. Examples of a useful mimetibody include butare not limited to EPO mimetibody, Remicade mimetibody, BMP mimetibody,cartilage derived morphogenic protein (CDMP) mimetibody and MP-52. In apreferred embodiment, the antibody is the EPO mimetibody.

In yet another embodiment, the therapeutic agent is a growth factor. Inan exemplary embodiment, the growth factors include, but are not limitedto, epidermal growth factor, bone morphogenetic protein, vascularendothelial-derived growth factor, insulin-like growth factor (IGF),hepatocyte growth factor, platelet-derived growth factor, hematopoieticgrowth factors, heparin binding growth factor, peptide growth factors,and basic and acidic fibroblast growth factors. In some embodiments itmay be desirable to incorporate genes for factors such as nerve growthfactor (NGF), muscle morphogenic factor (MMP), or TGF-beta superfamily,which includes BMPs, CDMP, and MP-52. In yet another embodiment, thetherapeutic agent is a receptor. Examples of receptors include, but arenot limited to, EPO Receptor, B Cell Receptor, Fas Receptor, IL-2Receptor, T Cell Receptor, EGF Receptor, Wnt, Insulin Receptor, TNFReceptor.

In other examples, the genetically modified chondrocyte can be used toexpress a therapeutic agent associated with a blood disorder, e.g., theEPO protein, by delivering the genetically altered chondrocyte to theliver or kidney, or any tissue or organ with a vascular supply to allowEPO to reach the target site or region. Once the EPO protein isexpressed, it can enter the circulatory system and bind to, and alterthe function of the EPO receptor (EPOR). This in turn will cause achange in the environment associated with the EPO receptor, for exampleby modifying the signal transduction cascade involving the EPO receptor.

Modification of the tissue may occur directly, for example by overexpressing EPO in a target region. Alternatively, modification of atissue may occur indirectly, for example by the over expressed EPOinteracting with an EPOR that leads to changes in downstream signaltransduction cascades involving the EPOR. Non-limiting examples ofmodifications include cell proliferation response, cell differentiation,modifications of morphological and functional processes, under- orover-production or expression of a substance or substances by a cell,e.g., a hormone, growth factors, etc., failure of a cell to produce asubstance or substances which it normally produces, production ofsubstances, e.g., neurotransmitters, and/or transmission of electricalimpulses.

As mentioned, the delivery device must be initially delivered to thetreatment site. The device can be implanted subcutaneously through asimple incision in the skin. Alternatively, the device can be surgicallyimplanted into a target region using standard surgical methods such asopen surgery, or more preferably by minimally invasive surgical methods,such as by using a trocar.

In general, the treatment site can be any site wherein a therapeuticagent can provide a therapeutic benefit. In an exemplary embodiment, thetreatment site is an atypical chondrocyte environment (i.e., anenvironment not usually associated with chondrocytes). Examples of anenvironment not usually associated with chondrocytes include the centralnervous system (CNS), which includes the brain and spinal cord. Otherexamples of environments that are not usually associated withchondrocytes include solid organs. Examples of solid organs include, butare not limited to, the heart, kidney, liver and pancreas. Yet anotherexample of an environment not usually associated with chondrocytes arethe reproductive organs. In males, the reproductive organs notassociated with chondrocytes are, for example, the testis, vas deferens,and the like. In females, the reproductive organs not associated withchondrocytes are, for example, the uterus, fallopian tubes, ovaries andthe like. Other examples of an environment not associated withchondrocytes include the blood, plasma, cerebrospinal fluid (CSF), skin,a subcutaneous pouch, intramuscular and intraperitoneal space.

The following examples are illustrative of the principles and practiceof this invention. Numerous additional embodiments within the scope andspirit of the invention will become apparent to those skilled in theart.

EXAMPLES Example 1 In Vitro Isolation and Culturing of Chondrocyte Cells

This example describes one of many possible methods of isolating andculturing chondrocyte cells. Articular cartilage was asepticallyobtained from calf knee joints. Cartilage was shaved from the end of thebones using a scalpel. The tissue was finely minced in saline solutionand submitted to collagenase and trypsin digestion for several hours at37° C. until the tissue was completely digested and single cells weregenerated. The chondrocyte cells isolated from the cartilage tissue werethen washed from enzyme and seeded onto tissue culture plastic dishes ata concentration of about 10,000 cells/cm². The chondrocyte cells whereexpanded in 10% FBS containing DMEM culture medium. Following expansion,the chondrocytes can be released from the vessel using a trypsin/EDTAtreatment, centrifuged and concentrated or diluted to a desiredconcentration to achieve for example a seeding density of approximately5,000 cells to about 10,000 cells per square centimeter when plated ontoculture dishes. Alternatively, the expanded chondrocytes cells can befrozen according to standard procedures until needed.

Example 2 In Vitro Transfection of Chondrocyte Cells and Expression of aMarker Protein

This example described the techniques used to transfect chondrocyteswith a marker protein, Green fluorescent protein (GFP). A DNA vectorcarrying a reporter gene is used to establish proof of concept for theutility of normal articular chondrocytes as a drug delivery system fortherapeutic application in ectopic sites. As a model, a DNA vector(pTracer-SV40) carrying a gene for green fluorescent protein was used asa surrogate to model a protein not normally present in chondrocytes, andof therapeutic value. If chondrocytes can be transfected by the modelvector and expressed the foreign protein, it would demonstrated theability of using this system for therapeutic protein. In the followingexample, a lipid formulation from Stratagene (GeneJammer) was used tointroduced the foreign DNA into chondrocytes. Any other reagent orphysical means of introducing DNA into cell would serve the samepurpose.

Exponentially growing chondrocytes were seeded onto 35 mm dishes at adensity of 1.5 to 3.7×10⁶ cells in 10% FBS containing DMEM. In separateplastic tube, serum-free medium was combined with transfection reagent(GeneJammer from Stratagene) and incubated with the chondrocytes for5-10 minutes. Plasmid DNA (pTracer-SV40 from Invitrogen) was mixed withthe chondrocytes, and the mixture further incubated for 5-10 minutes.The transfection mixtures was then added to the 35 mm dishes andincubated for at least 3 hours at 37° C. Fresh media (10% FBS containingDMEM) was then added and the cells are fed every 2-3 days with freshserum containing media.

In order to identify transfected chondrocytes, the culture chondrocytecells were observed under fluorescent light. To facilitate this, theculture media was removed and replaced with phosphate buffered salinesolution. The cultured chondrocyte cells were examined by fluorescencemicroscopy. Positive chondrocyte cells were identified as those thatincorporated the plasmid, expressed GFP, and fluoresced green under themicroscope, as shown in FIG. 12.

The results illustrate the ability of introducing and expressing aforeign protein in articular chondrocytes. These results further showthat chondrocytes provide a robust, stable cell for expression of aheterologous protein.

Example 3 In Vitro Transfection of Human and Bovine Chondrocyte Cellsand Expression of EPO Protein

This example describes the techniques used to transfect chondrocyteswith a therapeutic agent, human erythropoietin (EPO).

An EPO vector was generated which comprised the EPO gene (GenbankAccession No. 182198) inserted into the commercially available pSG5backbone (Stratagene Inc.). The pSG5 plasmid contains ampicillin andkanamycin as a selection marker for bacteria. This construct alsocontains F1 filamentous phage origin and SV40 promoter for betterexpression. The 584 base pair EPO fragment was cloned into the NCO/BamH1site site of pSG5. The size of this vector was about 4.6 kb.

A second EPO vector was generated which comprised the EPO gene insertedinto the commercially available pcDNA3.1 backbone (Invitrogen LifeTechnologies). This particular construct has the antibiotic Neomycin asa selection marker for mammalian cells.

Each of the EPO vectors was transfected into human chondrocytes usingFuGENE 6, a commercially available lipid transfection agent (RocheDiagnostics Corporation). Primary human chondrocytes were passaged once,then seeded onto 35 mm plates at a density of 250,000 cells/plate in 10%FBS containing DMEM. The transfection reagents were combined in serumfree medium at a ratio of 3 μl FuGENE to 1 μg plasmid and incubated for15 minutes at room temperature. The resulting transfection mixture wasthen added dropwise to the chondrocyte plates, with a total of 1.0 μg ofEPO plasmid added to each plate. The cell cultures were incubated at 37°C. Supernatant was collected, in aliquots of 0.5 ml, at 20, 48, 72 and96 hours and stored at 4° C.

For this experiment, two control samples were prepared. The firstcontrol comprised chondrocytes which were transfected with a geneencoding an EPO mimetibody. The second control comprised cells to whichno reagents were added. The above transfection procedure was repeatedfor human and bovine chondrocytes, with similar cell density, reagentconcentrations and control conditions.

Expression of EPO was determined by measuring the supernatant EPOconcentration synthesized by the chondrocytes using a commerciallyavailable ELISA kit which is specific to EPO (Quantikine IVD, R&DSystems Inc.). Absorption at 450 nm was measured to determine the amountof bound protein on the ELISA plates.

FIGS. 13 a and b shows that, for both human and bovine chondrocytes,neither control samples exhibited significant EPO expression, while bothof the EPO transfection constructs resulted in significant EPOexpression. Expression of human EPO by both human and bovinechondrocytes demonstrates the feasibility of allogeneic and xenogenicapproaches to chondrocyte-based in vivo therapeutic delivery.

Example 4 In Vitro Transfection of Human Chondrocyte Cells andExpression of EPO Mimetibody

This example described the techniques used to transfect chondrocyteswith a therapeutic agent, the EPO mimetibody (mEPO). This compound isdesigned to mimic the binding region of the EPO protein, therebyproviding the same therapeutic function as EPO when it attaches to theEPO receptor and activates it.

A construct was developed comprising the mEPO gene inserted into thecommercially available pcDNA3.1 backbone (Invitrogen Life Technologies).This particular construct has the antibiotic, Neomycin as a selectionmarker for mammalian cells, and Ampicillin as a selection marker forbacteria. The size of this vector construct was about 6 kb. Anotherconstruct was developed comprising the mEPO gene inserted into thecommercially available pCEP4 backbone (Invitrogen Life Technologies).This construct has hygromycin as a selection marker for mammalian cells,and contains EBV replication origin and nuclear antigen (EBNA-1) forbetter expression. The size of this vector is about 11 kb.

Each plasmid encoding mEPO was transfected into human chondrocytes usingthe method outlined in Example 3. Control samples comprised chondrocytestransfected with human EPO, as well as cells to which no reagents wereadded.

Expression of mEPO was measured using an ELISA which is specific to thehuman IgG region of the mimetibody. After 96 hours of transfection, EPOmimetibody expression was detected at a level of 18 ng/ml. As shown inFIG. 14, this expression level was substantially higher than thebaseline mEPO level measured for control samples. Collectively, theseresults demonstrate that chondrocytes can be used to express atherapeutic agents such as EPO and mEPO.

Example 5 Creating Chondrocyte-Gel Substrates

This Example describes how to formulate biological gel matrix substratescomprising genetically altered chondrocytes that express a therapeuticagent. Chondrocytes growing in monolayer (FIG. 15A) were released fromthe culture dish using trypsin/EDTA. The cells were counted andsuspended in low melting agarose at 40° C. prepared in serum free mediumsuch that the final concentration of cells was 100,000/ml of agarosegel. 3 ml of gel was dispensed into a 30 mm dish and allowed to set atroom temperature. The gel was then overlayed with an equal volume of 10%FBS containing culture medium. The media is replenished every 2 to 3days and cultures maintained for several weeks. After a few weeks,colonies of differentiating chondrocytes can be observed (FIG. 15B). Thegels were fixed in formalin 6 weeks after initial suspension and stainedwith alcian blue. Cartilage matrix deposited by the chondrocytes stainsblue and indicates cartilage differentiation (FIG. 15C). Monitoring oftherapeutic protein expression could be performed by sampling theculture media overtime. Establishing a delivery dose from such agarosesuspended chondrocytes could be done by harvesting different size disksgenerated for example with a biopsy punch.

Example 6 Use of Chondrocytes to Express a EPO or mEPO in a Non-TypicalChondrocyte Environment for a Blood Disorder

This example describes the use chondrocytes that have been geneticallyaltered to express EPO or mEPO in an in vivo system. The chondrocyteswere genetically altered as described in Examples 3 and 4 to express EPOor mEPO, and placed into biological gel matrix substrates as describedin Example 5. Slices of the solid gel, or a liquid gel that solidifieson contact, can be placed into an animal model of anemia (See Osborn etal, Supra), and the amelioration of anemia was observed by measuring thehemocrit levels of the blood.

Example 7 Use of Chondrocytes to Express Therapeutic Agent in aNon-Typical Chondrocyte Environment for an Autoimmune Disorder

This example describes the use chondrocytes that have been geneticallyaltered to express a therapeutic agent for amelioration of an autoimmunedisorders, such as expressing insulin an animal model for diabetes.

The chondrocytes were genetically altered as described in Example 2 toexpress the insulin gene, and placed into biological gel matrixsubstrates as described in Example 5. Slices of the solid gel, or aliquid gel that solidifies on contact, can be placed (e.g., into thepancreas) in an animal model of insulin (See e.g., Wong et al. Supra),and changes in the blood glucose levels can be observed.

Example 8 Use of Chondrocytes to Express a GABA in a Non-TypicalChondrocyte Environment for Neurodegenerative Disorder

This example describes the use chondrocytes that have been geneticallyaltered to express GABA an animal model for Parkinson's disease. Thechondrocytes were genetically altered as described in Example 2 toexpress GABA, and placed into biological gel matrix substrates asdescribed in Example 5. Slices of the solid gel, or a liquid gel thatsolidifies on contact, can be placed into a region of the brain (e.g.,subthalmic nucleus) in a model for Parkinson's disease (See e.g.,Bjorklund et al., Supra), and the amelioration of Parkinson's symptomscan be observed be monitoring the behavioral changes in the animal(e.g., by a maze test).

Example 9 Chondrocytes Contained in Agarose Gel

This example demonstrates the superior ability of chondrocytes tosurvive in large devices. Three different cell types, chondrocytes,ligament cells and dermal fibroblasts, were encapsulated in largealginate capsules. Cells at a density of 10⁶ cells were encapsulated incylindrical alginate gel plugs (Alginic acid sodium salt, Fluka # 71238)1 cm diameter×2 cm length and kept in either Dulbecco's Modified EagleMedium (DME or DMEM; Gibco/Invitrogen # 11995-040) with 10% fetal bovineserum (FBS) or DME/ITS (ITS—Insulin, Transferrin,Selenium—Gibco/Invitrogen # 51500-056) media for several weeks at 37° C.The cells were then stained with a live/dead dye (Molecular Probes #L3224) that stains the live cells in green and the dead cells in red.

A qualitative visual assessment of the results (see FIGS. 8A-10B) showthat the chondrocyte cultures have a higher proportion of green cellsthan red cells demonstrating their enhanced survivability in largecylindrical constructs.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

1. A method for delivering a therapeutic agent to a target regioncomprising: providing a housing configured to retain a geneticallyaltered chondrocyte and allow passage of the therapeutic agent, thehousing comprising: (i) a cell chamber; (ii) an auxiliary fluidcontainer to house an auxiliary fluid; and (iii) an expandable chamber;the auxiliary chamber being positioned between the cell chamber and theexpandable chamber; providing a genetically altered chondrocyte retainedwithin the housing, the genetically altered chondrocyte altered toexpress the therapeutic agent; delivering the housing and geneticallyaltered chondrocyte into the target region, wherein the geneticallyaltered chondrocyte expresses the therapeutic agent and the therapeuticagent is released from the housing in the target region, and wherein thetarget region is in an atypical chondrocyte environment.
 2. The methodof claim 1, wherein the atypical chondrocyte environment is in an organselected from the group consisting of the brain, heart, liver, kidney,gastro-intestinal tract, spleen, smooth and skeletal muscle, eye,ganglions, lungs, gonads, and pancreas.
 3. The method of claim 1,wherein the atypical chondrocyte environment is an aqueous environmentselected from the group consisting of blood and plasma.
 4. The method ofclaim 1, wherein the therapeutic agent is associated with a disorderselected from the group consisting of a blood disorder, an autoimmunedisorder, a hormonal disorder, an anti-inflammatory disorder, afertility disorder, and an neurodegenerative disorder.
 5. The method ofclaim 1, wherein the housing comprises a rigid material.
 6. The methodof claim 1, wherein the housing further includes at least onesemi-permeable port configured to allow passage of the therapeuticagent.